The broad objective is to reveal the molecular mechanisms by which erythropoietin (Epo) is degraded and cleared from the body, and to understand the role that these mechanisms of Epo degradation play in red blood cell homeostasis. The hypothesis is that binding of Epo to its receptor (EpoR) and subsequent endocytosis are essential for Epo degradation, and therefore the receptor binding and trafficking properties of Epo control its degradation and clearance from the body. To test this at the cellular level, the degradation rate of Epo or mutant Epo molecules when incubated with cells expressing the EpoR or mutant EpoR will be measured. The kinetics of receptor binding and ligand trafficking of Epo and mutant Epo molecules will be measured and analyzed with a mathematical model. To test the hypothesis in vivo, the clearance rate from serum and tissue distribution of Epo or mutant Epo molecules will be measured in wild-type mice and in mice with the EpoR expressed only in specific tissues or at a lower level. Since EpoR signaling is required for production of red blood cells, the rate at which the Epo ligand is degraded plays a role in red blood cell homeostasis and also will influence the effectiveness of Epo ligands injected in medical practice.